Abstract: This dissertation is devoted to studying the structure-function relationships of proteins using the methods of molecular modeling. In the opening chapter, the emphasis is on the theoretical basis of protein stability. Stability is one of the most important properties and the use of proteins depends on good stability in both basic research and industry. Stability can be divided into thermodynamic (the energy difference between folded and unfolded state) and kinetic stability (separation of relevant states by high activation energy). Several possibilities of protein stabilization are discussed ranging from purely experimental methods based on directed evolution and saturation mutagenesis, through identification of hot-spots, to sophisticated algorithms combining free energy calculations with the evolutionary inference. Next two chapters in introduction deal with the molecular modeling methods and model proteins used in the results section. Molecular dynamics is one of the most important methods that can be used to describe protein stability, activity, folding, hydration, enantioselectivity and substrate specificity. The other methods are molecular docking, which predicts binding modes and binding energy of the substrate-enzyme complex, and hybrid quantum mechanics and molecular mechanics methods used to describe the reactivity of macromolecules with small molecules. Results section consists of five parts. The first one (chapter 4), deals with the computational method FireProt applied for effective stabilization of a protein. FireProt combines evolutionary approach with a calculation of Gibbs free energy, complemented by efficient filtering using a variety of in silico tools. The method was applied to the enzyme haloalkane dehalogenase DhaA and dehydrochlorinase LinA resulting in thermostability increase 24 and 21 °C, respectively. Chapter 5 is focused on the stabilization of human fibroblast growth factor FGF2. This protein is involved in numerous regulatory processes, suggesting a good applicability in both medicine and basic research. Nowadays, the main application of FGF2 is its addition into the medium used for stem cells cultivation. However, FGF2 is not very stable with a half-life between 10 and 12 hours. Stabilizing mutations were identified by using the free energy calculation by Rosetta and FoldX in combination with evolutionary approach "back-to-consensus". These hits were combined with mutations from saturation mutagenesis libraries. The final nine-point mutant showed thermostability increase of 19 °C and was stable in the cultivation medium for more than twenty days. The chapters 6-8 deal with a characterization of DhaA enzyme. Chapter 6 focuses on the stability-activity relationships. A stable four-point mutant of DhaA showed tolerance against organic co-solvents, but on the other hand had a very low activity. A single mutation at the mouth of access tunnel significantly improved activity while preserving the thermostability and stability against organic co-solvents. A single mutation increased the mobility of secondary structures and significantly increased the diameter of the access tunnel. Chapter 7 deals with a method for the analysis of protein hydration. A fluorescent artificial amino acid, which provides a different signal in the presence of varying number of water molecules, was introduced into the tunnel mouth of two dehalogenases. Using molecular dynamics, different hydration of both dehalogenases was observed, which is in close correlation with experimental fluorescent spectroscopy. The last 8th chapter is devoted to the study of enantioselectivity of dehalogenases. Previous studies showed that the enantioselectivity of DbjA dehalogenase with 2-bromopentane is due to increased hydration in the accessible active site. Active site hydration aligned the substrate with the hydrophobic wall of the protein favoring the conformation of (R)-enantiomer. A five-point mutant of DhaA has the opposite properties of

Abstract: its active site than DbjA (less hydrated and less accessible active site) and yet exhibits the same enantioselectivity. The difference in enantiodiscrimination of dehalogenases was explained using a combination of site-directed mutagenesis, kinetic measurements, and molecular modeling.